Constitutive activation of the MAP kinase pathway is a common event in human tumors and activating mutations in this pathway occur in RAS, BRAF and upstream receptor tyrosine kinases (RTK) in a mutually exclusive fashion. We find that tumor cells with activating BRAF(V600E) mutations are selectively sensitive to PD0325901, a selective inhibitor of MEK1/2. Tumors in which MAPK is activated by other upstream events (mutant RAS, RTK activation) are typically less sensitive or resistant to MEK inhibition. The primary objective of this proposal is to identify genetic predictors of sensitivity and resistance to inhibitors of the RAF/MEK pathway. Three aims are proposed. In Aim 1, we will compare pharmacologic inhibition of RAF, MEK and siRNA knockdown of these targets. These studies will serve to validate the in vivo selectivity of the RAF selective inhibitor PLX4032 and determine whether oncogenic BRAF promotes cell survival via effectors other than MEK kinase. The possibility that BRAF mutations promote tumorigenesis through MEK-independent effectors has important implications. If all of the oncogenic effects of mutant BRAF are mediated through MEK/MAPK, MEK and pan-RAF inhibitors should be equally effective. However, if important effects of BRAF are mediated by other effectors, RAF inhibitors would be predicted to be superior. Our preliminary data also suggests that the purely cytostatic response of some BRAF mutant tumors to MEK inhibition and the resistance of many RAS mutant tumor to MEK inhibition may be the result of additional genetic alterations in parallel signaling pathway such as the PI3K/AKT pathway. BRAF mutations commonly co-exist with PTEN loss in melanoma. Therefore, in Aim 2, we will determine whether loss of PTEN function is sufficient to convert the response of V600E BRAF mutant tumors to MEK (and RAF) inhibition from a cytotoxic to a cytostatic response. Finally, in Aim 3, we will determine in tumors with RAS mutations whether concurrent p1101 PI3 kinase (PIK3CA) mutations confer resistance to RAS knockdown (by shRNA) or MEK inhibition. If PTEN loss or PIK3CA mutations are sufficient to confer resistance of BRAF and RAS mutant cell to RAF/MEK pathway inhibition, we will test combinations of RAF/MEK and PI3K/AKT pathway inhibitors in such tumors. The long-term goal of these efforts will be to accelerate the development of MEK and RAF inhibitors by identifying the genetic profile of those tumors most likely to respond. Further, by identifying genetic predictors of resistance to RAF and MEK inhibitors, the studies outlined would provide a molecular basis for studies of rational combinations of signaling inhibitors tailored to patients whose tumors have mutations in both the RAS/RAF and PI3k/Akt pathways. PUBLIC HEALTH RELEVANCE: RAS and BRAF mutations are among the most common genetic alterations in human tumors and cancers expressing these mutations respond poorly to standard cytotoxic chemotherapies. The primary goals of this proposal are to identify genetic predictors of sensitivity and resistance to inhibitors of the RAS/RAF/MEK pathway and use this information to develop novel strategies for cancer therapy.